ZO‐1 Knockdown in MDCK Type II Cells Decreases Leak Pathway Opening Size and Increases Opening Number

Abstract

Paracellular solute movement across epithelial cell sheets is divided into at least two pathways, the Pore Pathway (high capacity, small ions and solutes, charge selective) and the Leak Pathway (low capacity, large molecules, charge insensitive). Improved understanding of the Leak Pathway is essential to evaluate its role in epithelial physiology and pathophysiology, as well as, its potential use as a pathway for the delivery of macromolecule drugs. Depletion of the tight junction‐associated cytoplasmic protein Zonula Occludens‐1 (ZO‐1) in the dog kidney epithelial cell line, MDCK Type II, increases Leak Pathway apparent permeability (Papp). There are multiple parameters that could affect Leak Pathway Papp, including the density of the pores/openings (number/unit membrane area), their length, their tortuosity, and their size (radius). A theoretical computational analysis revealed marked differences in the effect of pore size versus pore density on Papp­ as a function of solute size. We, therefore, compared the dependence of Papp on solute size in populations of wild type MDCK Type II cells, ZO‐1 knockdown MDCK Type II (ZO‐1 KD) cells, and ZO‐2 knockdown MDCK Type II (ZO‐2 KD) cells. The flux of fluorescein‐dextrans of different sizes across monolayers of wild type MDCK Type II, ZO‐1 KD, and ZO‐2 KD cell populations grown on permeable supports was measured as a function of time. Flux was linear as a function of time for all solutes across all cell lines. As expected, flux rate decreased with increasing fluorescein‐dextran Stokes radius (3 kDa ‐ ~12 Å to 70 kDa ‐ ~60 Å) in all three cell lines. For the smaller fluorescein‐dextrans, ZO‐1 KD cell populations exhibited several‐fold higher Papp values than wild type MDCK Type II cells. As solute size increased, however, the extent of the increase over the wild type MDCK Type II cells diminished progressively. In contrast, the Papp values for the wild type MDCK Type II cell populations and the ZO‐2 KD cell populations were similar throughout the fluorescein‐dextran size range. These results revealed that Papp values decreased more rapidly as a function of solute size in ZO‐1 KD cell populations than in wild type MDCK Type II cell populations or ZO‐2 KD cell populations. While we cannot determine Leak Pathway opening radius using this approach, our results argue that the Leak Pathway openings in ZO‐1 KD cell populations are smaller than those in either wild type MDCK Type II cell populations or ZO‐2 KD cell populations. Since the ZO‐1 KD cell populations exhibit an increased flux rate for smaller fluorescein‐dextrans, this suggests that the ZO‐1 KD cell populations have an increased number of Leak Pathway openings compared to wild type MDCK Type II cell populations and ZO‐2 KD cell populations. Differences in opening length and/or tortuosity may also play a role in the observed differences in solute flux rates.